Peroxide/essential oils containing mouthwash compositions and two-part mouthwash systems

ABSTRACT

An antimicrobial mouthwash composition that is useful in the prevention and reduction of bad breath, plaque and related gum diseases comprises an antimicrobial effective amount of thymol and one or more other active essential oils; optionally, ethanol in an amount up to about 30% v/v; from about 0.1% to about 8.0% w/v of a peroxide; at least one surfactant in an amount sufficient to solubilize the essential oils; and water. The compositions may be reduced alcohol or alcohol-free compositions. Further a two-part mouthwash system is disclosed, comprising a base composition and a peroxide solution which may be mixed to form an antimicrobial mouthwash composition. The actives not only provide enhanced efficacy but are completely solubilized, thus providing an aesthetically appealing product.

CROSS REFERENCE

This application is a national stage application under Title 35, UnitedStates Code 371 of PCT/US97/00439 filed Jan. 15, 1997, which claimspriority under Title 35, United States Code 119(e) to ProvisionalApplication Ser. No. 60/010,508, filed Jan. 24, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to mouthwashes for the prevention andelimination of bad breath as well as for the reduction of oralmicroorganisms resonsile for the development of dental plaque and toothdecay. Dental plaque can lead to the formation of calculus, gingivitisand other related gum diseases. In particular, the present inventionrelates to a mouthwash which is effective in preventing these problemsand which contains both hydrogen peroxide and one or more essential oils(hereinaer referred to as “a hydrogen peroxide/essential oils containingmouthwash”. The mouthwash may be a high alcohol mouthwash (e.g.,containing up to about 25-30% by volume of alcohol), a reduced alcoholmouthwash or an alcohol-free mouthwash with the reduced alcoholmouthwash being particularly preferred. The invention also relates to atwo-part mouthwash system for delivering and preparing the mouthwashesof this invention.

2. Description of Related Art

Oral rinse and mouthwash compositions have been used by people for manyyears for the prevention of bad breath and for the elimination ofbacteria and other oral microorganisms that are responsible not only forbad breath but also tooth decay, plaque and gum diseases such asgingivitis and periodontitis. To this end, antiseptic mouthwashes in thepast have been designed to clean the oral cavity, provide fresh breathand kill these pathogenic microbes.

Thymol, a well known antiseptic compound also known as an essential oil,is utilized for its antimicrobial activity in a variety of mouthwashpreparations. In particular, thymol can be utilized in oral hygienecompositions such as mouth rinses in sufficient quantities to providedesired beneficial therapeutic effects. Listerine®-brand mouthwash is awell-known antiseptic mouthwash that has been used by millions of peoplefor over one hundred years and has been proven effective in killingmicrobes in the oral cavity that are responsible for plaque, gingivitisand bad breath. Thymol, together with other essential oils such asmethyl salicylate, menthol and eucalyptol, are active ingredients inantiseptic mouth rinses such as Listerine®. These oils achieve theirefficacy although present in small amounts. Without being restricted toany specific theory, it is now believed that the efficacy and taste ofantiseptic mouthwashes such as Listerine® may be due to the dissolutionof these four active ingredients. Dissolution is also important from anaesthetic point of view since a clear mouthwash solution is certainlypreferred by consumers to one that is cloudy, turbid or heterogeneous.

The leading antiseptic mouthwashes have always contained alcohol (i.e.,ethanol) at fairly high levels, ranging from approximately 25 up toabout 30% by volume, based on the total mouthwash volume (hereinafterreferred to as “% v/v”). Alcohol is used both as a vehicle and as asolvent in which the active ingredients, and additives such asastringents, fluorides, color additives, flavor oils, and the like, canbe dissolved and then dispersed into solution. Alcohol also provides apreservative role for the mouthwash during storage and use, and enhancesthe flavor oil organoleptic cues.

However, the use of high levels of alcohol has been recently challengedfrom an overall health standpoint, even though clinical data have notproven adverse health risk effects.

Merely reducing the levels of alcohol in these mouthwash compositions,however, has significant disadvantages. It has been found that loweralcohol levels result not only in a loss in the solubility of theactives and other ingredients in the composition, but there is also anoticeable decrease in the ability of the composition to kill the oralmicroorganisms responsible for bad breath, plaque and gum disease.

Hydrogen peroxide has been employed in alcohol and alcohol-freemouthwash compositions. While hydrogen peroxide is a known bactericide,it is not universally antiseptic and therefore its anti-plaque efficacyis suspect. Thus, a mouthwash composition containing anti-plaqueeffective essential oils and hydrogen peroxide which results in improvedanti-plaque efficacy over compositions containing essential oils alonewould be unexpectedly advantageous. Moreover, there is a substantialneed for the development of a reduced alcohol mouthwash in which theessential oils are completely dissolved and that continues to beeffective in the prevention of bad breath the killing of oral microbesand the resultant reduction or elimination of plaque.

The prior art reflects a number of attempts at formulating hydrogenperoxide-containing reduced alcohol and alcohol-free mouthwashcompositions. For example, U.S. Pat. Nos. 5,310,546 and 5,174,990disclose a mouthrinse formulation having between about 0.25% and about0.65% by weight, based on the total volume of the composition(hereinafter referred to as “% w/v”), of hydrogen peroxide, betweenabout 0.005% and about 0.1% w/v zinc chloride, at least about 0.03% w/vsodium lauryl sulfate, at least about 0.006% w/v citric acid, at leastabout 0.012% w/v sodium citrate and less than about 5% ethanol. Theethanol may be denatured with denaturing agents, generally in an amountless than 0.1% of the mouth rinse. The disclosed denaturing agentsinclude anethole, anise oil, bay oil, bergamot oil, bitter almond oil,cedar leaf oil, cinnamon oil, clove oil, eucalyptol, eucalyptus oil,eugenol, lavender oil, menthol, peppermint oil, sassafras oil, spearmintoil, terpeneless spearmint oil, thyme oil, thymol and wintergreen oil,alone or in combination.

U.S. Pat. No. 4,900,721 discloses an aqueous disinfectant fordisinfecting the skin and mucous membranes. The disinfectant maycomprise 8 to 25% by weight ethanol, based on the total weight of thecomposition (hereinafter referred to as “% w/w”), 0.2 to 0.7% w/whydrogen peroxide, 0.1 to 0.5% w/w of at least one carboxylic acid, suchas lactic acid or benzoic acid, 0.05 to 1.0% w/w of at least onemicrobicidally active nitrogen-containing organic compound, such aschlorhexidine gluconate, 0.01 to 0.2% w/w of a microbicidally activecompound, such as an ethereal oil containing 91.4% peppermint oil (90%menthol, 4.0% salicylic acid phenylester, 3.5% anethole, 0.6% eugenoland 0.5% thymol) and the balance water. This reference alleges thatantimicrobial phenolic compounds produce a synergistic antimicrobialeffect when combined with the other compounds. The reference alsodiscloses adding an emulsifier at a ratio of 2:1 to 1:2 of the phenoliccompound. The microbicidally active, nitrogen-containing organiccompound is an essential component of the composition.

U.S. Pat. No. 4,431,631 discloses an oral aqueous solution containing 1to 3% w/v hydrogen peroxide, 3-15% w/v of a polyhydric alcohol, such asglycerin or sorbitol, 3-10% w/v ethanol, 0.3 to 2% w/v of a nonionicwater soluble polyoxyethylenated polyoxypropylene polyol surfactant, 0.3to 2% w/v of a nonionic surface active water soluble polyoxyethylenatedmonester of sorbitol with C₁₀₋₁₈ fatty acid, a sweetener compound and aflavor selected from either (i) a wintergreen flavor containing methylsalicylate and menthol in a weight ratio of about 3:1 to 5:1 and (ii) acinnamon flavor that is a propylene glycol solution containing about6-9% menthol, 32-38% cinnamic aldehyde and 6-9% clove oil. Thisreference alleges that the specifically disclosed flavorants are notadversely affected by the hydrogen peroxide. In U.S. Pat. No. 4,537,778,an aqueous oral composition containing only 1 to 3% w/v hydrogenperoxide and the particular above-described flavorant is disclosed, butnot exemplified. This patent describes the other components listed inU.S. Pat. No. 4,431,631 as preferred adjuvants.

U.S. Pat. No. 5,104,644 discloses a mouthrinse preparation having about0.5 to about 3% w/v of hydrogen peroxide, at least 0.02% w/v zincchloride, at least about 0.04% w/v sodium lauryl sulfate, at least about0.08% w/v sodium citrate and about 2 to 3.5% w/v ethanol. Peppermint oiland menthol are used as flavorants. The reference alleges that thecomposition is capable of killing bacteria associated with dentaldisease.

U.S. Pat. Nos. 5,302,373 and 5,330,749 disclose a concentrated aqueousethanolic mouthwash formulation comprised of 5 to 50% w/w ethanol, 5 to35% w/w water, about 0.5 to 30% w/w of a humectant ingredient and about2 to 30% w/w of a suspension of particulate alkali met bicarbonateingredient with an average particle size of about 0.5-5 microns. Theconcentrate may be diluted with about 0.2 to 4 parts by volume water perpart by volume of concentrate. These references disclose that optionalbactericides may be selected from, among others, thymol or hydrogenperoxides and that optional flavorants may include, among others,peppermint, wintergreen, eucalyptus or methyl salicylate.

U.S. Pat. No. 5,200,194 is directed to an oral osmotic device fordelivering a drug into the mouth of a human patient The reference lists,among many others, ethanol, hydrogen peroxide and non-charged phenolicagents such as thymol as suitable anti-plaque agents. This referencedoes not suggest or disclose the combination of those agents,particularly in a mouthwash formulation.

Clearly, a hydrogen peroxide/essential oils containing mouthwash havinggreater anti-plaque efficacy that exhibited by mouthwashes employingessential oils alone would be both unexpected and significantlyadvantageous. Moreover, a need clearly exists for a reduced alcohol oralcohol-free composition that is highly efficacious in the prevention ofbad breath, plaque and gum disease. In addition, there is a need forsuch oral compositions that both kill the oral microflora responsiblefor these problems cleaning the oral cavity leaving a fresh, lubricousmouthfeel, and also cleaning teeth.

SUMMARY OF THE INVENTION

The present invention is a hydrogen peroxide/essential oils containingantimicrobial mouthwash composition with a high level of efficacy in theprevention of plaque, gum disease and bad breath, and, in particular,the invention is a reduced alcohol or alcohol-free mouthwashcomposition. In addition, the antimicrobial oral mouthwash compositionsof this invention are clear, aesthetically appealing products.

A high alcohol (up to about 30% v/v of ethanol) peroxide/essential oilscontaining mouthwash of this invention provides an effective anti-plaquemouthwash composition. However, it is highly preferred to employ thereduced alcohol (up to about 22% v/v of ethanol) mouthwash compositionor even the alcohol free (substantially free of ethanol) mouthwashcompositions of this invention.

In one embodiment, the present invention provides a hydrogenperoxide/essential oils containing antimicrobial mouthwash compositionwhich comprises: (a) an antimicrobial effective amount of thymol and oneor more other essential oils; (b) ethanol in an amount up to about 30%v/v; (c) from about 0.1% to about 8.0% w/v, preferably from about 0.1%to about 3.0% w/v, and most preferably from about 0.1% to about 2.9%w/v, of a peroxide; (d) a sufficient amount of at least one surfactantto assist in the solubilization of said essential oils; and (e) water.The R-Factor (a measure of anti-plaque efficacy, described infra) ofsaid compositions is generally less than about 2.0, preferably less thanabout 1.2, most preferably less than about 1.0.

A particularly preferred embodiment of the present invention provides areduced alcohol, antimicrobial mouthwash composition which comprises:(a) an antimicrobial effective amount of thymol and one or more otheressential oils; (b) ethanol in an amount of up to about 22% v/v,preferably no more than about 10% v/v; (c) from about 0.1% to about 8.0%w/v, preferably from about 0.1% to about 3.0% w/v, and most preferablyfrom about 0.1% to about 2.9% w/v, of a peroxide; (d) a sufficientamount of at least one surfactant to assist in the solubilization ofsaid essential oils; and (e) water. The preferred peroxide of thisinvention is hydrogen peroxide, although any peroxide or hydrogenperoxide precursor, e.g., sodium percarbonate, sodium peroxide, chlorinedioxide, other peroxide salts, or in combination may be employed.

In another embodiment, the present invention provides an alcohol-freeantimicrobial mouthwash composition which comprises: (a) anantimicrobial effective amount of thymol and one or more other essentialoils; (b) from about 0.1% to about 8.0% w/v, preferably from about 0.1%to about 3.0% w/v, and most preferably from about 0.1% to about 2.9%w/v, of a peroxide; (c) a sufficient amount of at least one surfactantto assist in the solubilization of said essential oils; and (d) water.This embodiment is substantially free of ethanol.

Another embodiment of this invention is directed to a peroxide/essentialoils containing two-part mouthwash system comprising: (i) a first vesselcontaining a base composition comprising thymol and one or moreessential oils, optionally ethanol, at least one surfactant and waterand (ii) a second vessel containing an aqueous peroxide solution.Preferably the resulting mouthwash composition is a reduced alcohol oralcohol-free composition. This two-part mouthwash system helps to ensurethe stability of the resulting mouthwash by isolating the othercomponents of the mouthwash from the hydrogen peroxide until just priorto use. The concentration of the components in the base composition andin the peroxide solution are such that upon mixing an amount of the basesolution with an amount of the peroxide solution, i.e., a predeterminedratio, a peroxide essential oils containing and preferably a reducedalcohol or alcohol-free mouthwash composition as described aboveresults. The amount of base solution that is mixed with the peroxidesolution, i.e., the ratio of the mixture, is predetermined so thatconcentration of the components in the base solution and aqueousperoxide solution can be adjusted to provide the inventive mouthwashcomposition upon mixing. This embodiment of the invention provides anadvantageous two-part mouthwash system which can be employed to deliverthe mouthwash composition of this invention without stability problemswhich may result from the mixture of a peroxide with the other activecomponents of the mouthwash composition.

DETAILED DESCRIPTION OF THE INVENTION

The mouthwash compositions of this invention provide an unexpected highdegree of antiseptic efficacy towards oral microorganisms responsiblefor oral malodor and the build-up of plaque and calculus and theresulting tooth and gum diseases that may follow. In particular, themouthwash compositions of this invention exhibit enhanced antimicrobialefficacy over compositions containing only essential oils or hydrogenperoxide alone. Significantly, these advantageous results are achievedfor the mouthwash compositions of this invention even with reducedalcohol content or when they are alcohol-free. Although the exactmechanism of action is unknown, enhanced antimicrobial efficacy isobtained when minor amounts of thymol and one or more other essentialoils (e.g., eucalyptol, menthol and methyl salicylate) are combined withabout 0.1% to about 8.0%, preferably about 0.1% to about 3.0% w/v of aperoxide and a surfactant in an amount sufficient to solubilize theessential oils. Most preferably the peroxide is employed in an amount offirm about 0.1% to about 2.9% w/v. The preferred peroxide is hydrogenperoxide. However, it is also possible to employ a hydrogen peroxideprecursor capable of generating hydrogen peroxide in solution. It isbelieved that the hydrogen peroxide synergistically enhances theantimicrobial efficacy of the essential oils.

Without being bound to theory, it is believed that the peroxide enhancesthe efficacy of the antimicrobial composition by causing pore channelformation and/or delipidization of bacterial membranes. It is furthertheorized that the peroxide may enhance the anti-plaque efficacy of thecomposition through hydrogen bonding, superoxide formation and/or asynergistic antimetabolic affect with the essential oils. Otherpotential mechanisms of action resulting in enhanced efficacy may beincreased peroxide tension in the saliva and plaque, improvedsubstantivity on the tooth and oral surfs and/or an increased diffusionof the essential oils. These potential mechanisms of action are setforth only as theory and do not in anyway limit the scope of thisinvention.

Thymol, (CH₃)₂CHC₆H₃(CH₃)OH (isopropyl-m-cresol), is only slightlysoluble in water but is soluble in alcohol, and its presence is one ofthe reasons alcohol was necessary in the well-established, high alcoholcommercial mouthwashes such as Listerine®. Methyl salicylate,(C₆H₄OHCOOCH₃), also known as wintergreen oil, additionally providesflavoring to the mouthwash together with its antimicrobial function.Eucalyptol (C₁₀H₁₈O; cineole) is a terpene ether and provides a cooling,spicy taste together with its antimicrobial functions. Menthol(CH₃C₆H₉(C₃H₇)OH; hexahydrothymol) also is only slightly soluble inalcohol, is fairly volatile, and in addition to any antisepticproperties provides a cooling, tingling sensation.

The essential oils are used in amounts effective to provideantimicrobial activity in the oral cavity. Generally, the total amountof essential oils present in a composition of this invention can be fromabout 0.001% to about 0.5% w/v, with about 0.16% to about 0.28% w/vbeing preferred. The compositions of the present invention containthymol and one or more other essential oils. Preferably the additionalessential oils are eucalyptol, menthol, or methyl salicylate, ormixtures thereof. Most preferably, the composition contains all four ofthese essential oils.

Thymol is preferably employed in amounts of from about 0.001% to about0.5% w/v, and most preferably from about 0.04% to about 0.07% w/v.Eucalyptol may be employed in amounts of from about 0.001% to about 0.5%w/v, and preferably from about 0.085% to about 0.10% w/v. Menthol ispreferably employed in amounts of from about 0.001% to about 0.5% w/vand most preferably from about 0.035% to about 0.05% w/v. Methylsalicylate is preferably employed in amounts of from about 0.001% toabout 0.5% w/v, and most preferably from about 0.04% to about 0.07% w/v.

The mouthwash compositions of this invention provide effectiveantimicrobial activity without the presence of other types ofantimicrobial compounds. For example, the compositions of this inventiondo not require known antimicrobial nitrogen-containing organiccompounds, such as chlorhexidine gluconate and the like, to achieveeffective antimicrobial activity. Thus, the reduced alcohol andalcohol-free peroxide containing mouthwash compositions of thisinvention are particularly advantageous over the compositions of theprior art.

Although not necessary, it is possible to employ other antimicrobialagents in the composition of this invention. Other exemplaryantimicrobial agents include triclosan, cetyl pyridium chloride,domiphen bromide, quaternay ammonium salts, zinc compounds, sanguinaninesoluble pyrophosphates, fluorides, alexidine, octonidine, EDTA, and thelike.

The carrier for the essential oils (i.e., the active ingredients) is anaqueous medium. The aqueous medium may be a water-alcohol-surfactantmixture, generally water-ethanol-surfactant. Alternatively, in the caseof alcohol-free compositions, the aqueous medium is a water-surfactantmixture. In the past, most antiseptic oral mouthwash compositions, suchas Listerine®-brand mouthwashes, required high ethanol levels of up toabout 27% v/v. These high levels were necessary to assist the actives inproviding the necessary antimicrobial functionality as well as providinga clear, aesthetically attractive liquid medium. Merely reducing thealcohol levels, without more, results in a cloudy, less efficaciousproduct.

Without being bound to any theory, it is believed that in these highalcohol level oral compositions, the alcohol solubilizes theantimicrobial actives and in so doing acts as an active enhancementmechanism. The actives are more readily dispersed throughout thesolution and can attack pathogenic microbes throughout the oral cavity.Reducing the alcohol levels was believed to adversely affect thisenhancement mechanism.

In accordance with a preferred embodiment of the present invention,however, it was surprisingly and unexpectedly found that alcohol may beused at reduced levels or may even be completely removed withoutsacrificing antimicrobial efficacy or clarity if the mouthwashcomposition also contains hydrogen peroxide and a surfactant. By“reduced level” of alcohol is meant an amount of ethanol up to about 22%v/v, preferably no more than about 10% v/v, and most preferably fromabout 0.1 to 10% v/v. By “alcohol-free” is meant that the composition issubstantially free of ethanol.

The hydrogen peroxide in combination with at least one surfactant hasbeen found to provide substantially equivalent and even enhancedantimicrobial activity of the essential oils for reduced alcohol andalcohol-free compositions compared to that exhibited by an antisepticmouthwash having high alcohol levels on the order of about 27% v/v. Aconvenient reference point is the antimicrobial activity exhibited by ahigh alcohol level Listerine®-brand mouthwash; i.e., one which containsabout 27% v/v ethanol. Expressed in terms of an “R-Factor,” which, asexplained below, represents the time necessary to effectively killtypical oral cavity microbes in an in vitro biofilm, normalized againstthe kill time exhibited in vitro by a 27% v/v Listerine®-brandmouthwash, the mouthwash compositions of this invention exhibit anR-Factor of less than about 2, preferably less than about 1.2, and mostpreferably less than about 1.0.

Surface active agents (surfactants) are employed in the compositions ofthis invention. They are organic materials which aid in the completedispersion of the ingredients throughout the solution as well asdispersing the preparation throughout the oral cavity. Preferably, thesurfactant used in the compositions of this invention is a non-ionicsurfactant or anionic surfactant employed in an amount sufficient tohelp solubilize the actives. Mixtures of non-ionic surfactant andanionic surfactant may also be used. By sufficient amount it is meantthat the surfactant is present in an amount that effectively assists inthe solubilization of the essential oils.

The preferred non-ionic surfactants are selected from the group known aspoly(oxyethylene)-poly(oxypropylene) block copolymers. Such copolymersare known commercially as poloxamers and are produced in a wide range ofstructures and molecular weights with varying contents of ethyleneoxide. These non-ionic poloxamers are non-toxic and acceptable as directfood additives. They are stable and readily dispersible in aqueoussystems and are compatible with a wide variety of formulations and otheringredients for oral preparations. These surfactants should have an HLB(Hydrophilic-Lipophilic Balance) of between about 10 and about 30 andpreferably between about 10 and about 25.

By way of example, non-ionic surfactants useful in this inventioninclude the following poloxamers:

105 188 237 334 108 215 238 335 124 217 284 338 184 234 288 407 185 235333

Generally these polymers when used are present in amounts of from about0.01% w/v to about 8.0% w/v, and preferably from about 0.25% to about0.75% w/v. A particularly preferred poloxamer is poloxamer 407 which isincorporated in an amount of about 0.1 to 4.5% w/v.

The preferred anionic surfactants are selected from sodium laurylsulfate, magnesium lauryl sulfate, zinc lauryl sulfate, Tauranol® andthe like. Tauranol® is sodium-N-methyl-N-cocoyl taurate available fromFinetex, N.J. Generally, the anionic surfactants when used are presentin amounts of about 0.01% to about 1% w/v, and preferably from about0.1% to about 0.5% w/v.

The surfactant is used to help solubilize the essential oils and flavoroils which may otherwise not be soluble in these aqueous systems due totheir reduced ethanol content. The surfactant(s) also act to dispersethe actives and flavors throughout the solution and enable thecompositions to provide a clear, uniform appearance that isaesthetically more appealing.

The compositions of this invention may contain a mixture of a non-ionicsurfactant and an anionic surfactant. An exemplary surfactant mixture,which is preferred, particularly when the composition is substantiallyfree of ethanol, includes poloxamer 407 and sodium lauryl sulfate.

In one embodiment of this invention, three types of peppermintoil—natural, Far West (redistilled, terpeneless) and Rose Mitchum—arecombined to provide a triple blend. This unique blend of flavor oils notonly provides a pleasant tasting mouthwash but also serves to taste maskthe bitter tasting essential oil actives discussed above. Each of thesepeppermint oils is present in substantially the same amount, from about0.001% w/v to about 1.0% w/v, and preferably in an amount of from about0.2% w/v to about 0.3% w/v. Combined, the triple blend is incorporatedin the mouthwash composition in an amount of approximately 0.01% w/v toabout 4.0 w/v, preferably in an amount of from about 0.5% w/v to about0.9% w/v.

The essential oil methyl salicylate not only provides antimicrobialaction but, being a wintergreen flavor oil, also adds to theorganoleptic flavor tones and complements the taste masking function ofthe peppermint oil blend.

Other flavor oils may also be added to further modify or magnify thecooling minty taste of the peppermint, or reduce or mask the sharp“bite” or “burn” of ingredients such as thymol and ethanol. Suitableflavors (and typical amounts) include spearmint oil (from about 0.01%w/v to about 1% w/v), oil of anise (from about 0.01% w/v to about 0.2%w/v), benzyl alcohol (from about 0.001% w/v to about 0.5% wv) andanethole (from about 0.01% w/v to about 0.5% w/v). Other flavors such ascitrus oils, vanillin, cooling compounds and the like may beincorporated to provide further taste variations.

The particular flavor oils and other taste-improving ingredientsemployed will vary depending upon the particular taste and feel desired.Those skilled in the art can select and customize these types ofingredients to provide the desired results.

Additional conventional components may be added as in mouthwashes of theprior art. Whereas some alcohol containing mouthwashes have a pH ofabout 7.0, reduction of the alcohol level, or its total elimination,requires the addition of preservatives which drops the pH tounacceptable levels. Buffer systems are then necessary to control the pHof the composition at optimal levels. This is generally accomplishedthrough the addition of a weak acid and its salt or a weak base and itssalt. Useful systems have been found to be sodium benzoate and benzoicacid in amounts of from about 0.01% to about 4.0% w/v, and sodiumcitrate and citric acid in amounts of from about 0.001% to about 0.2%w/v. Preferably the buffers are incorporated in amounts that maintainthe pH at levels of from about 3.5 to about 7.5, and more preferablyfrom about 4.0 to 5.0. Without being bound to any theory, it is believedthat these pH levels provide the essential oils with an environment thatalso maximizes their antimicrobial activity.

Other conventional ingredients may be used in the mouthwash compositionsof this invention, including those known and used in the art. Forexample, humectants such as polyethylene glycol may be added as anadditional solubilizer for the flavor oils and to also provide textureto the composition. These are incorporated in amounts of from about 0.3%w/v to about 0.6% w/v, and preferably about 0.5% w/v. Softeners such asglycerin may be added to enhance the lubricous mouthfeel of themouthwash as it is used and to provide a refreshing, moist, organolepticfeeling thereafter. Glycerin may be incorporated in amounts of fromabout 0.05% w/v to about 10.0% w/v, and preferably in an amount of about7.5% w/v. Sweeteners such as aspartame or sodium saccharin and the likemay be added for better taste in amounts of from about 0.005% w/v toabout 1.0% w/v, and preferably in an amount of approximately 0.05% wv.

Zinc chloride or other zinc salts e.g. zinc gluconate, zinc sulfateetc., may be added as an astringent for an “antiseptic cleaning” feelingin an amount of from about 0.0025% w/v to about 0.200% w/v. And althoughthe mouthwash formulations of this invention may be formulated to besubstantially clear and colorless, acceptably approved food dyes arepreferably used to provide a pleasing color to the formulations of theinvention. These may be selected from the long list of acceptable fooddyes and in particular may be incorporated to provide the spearmintgreen formulation discussed infra. Suitable dyes for this purposeinclude FD&C yellow #5, FD&C yellow #10 and FD&C green #3. These areadded in conventional amounts, typically in total amounts of from about0.0003% w/v to about 0.0005% w/v, and preferably from about 0.000035% wvto about 0.00045% w/v.

It is most preferable to prepare the mouthwash composition of thisinvention as a two-part mouthwash system. This is accomplished bypreparing a base composition having the above described components withthe exception of the peroxide. The peroxide is prepared as a separateaqueous solution. The concentration of the components in the basecomposition and the peroxide solution is dependent upon the ratio atwhich the base composition and peroxide will be mixed. The basecomposition and the peroxide solution are generally mixed at a ratiofrom about 1:10 to about 10:1 by volume. The preferred mixture ratio is1:1 by volume. Once the ratio has been determined it is a simplearithmetic calculation to determine the concentration of the componentsin the base composition and the peroxide solutions which will result inthe mouthwash composition of this invention after mixing at thepredetermined ratio.

The two-part mouthwash system of this invention is comprised of (i) afirst vessel containing a base composition comprising thymol and one ormore other essential oils, optionally ethanol, at least one surfactantand water and (ii) a second vessel containing an aqueous peroxidesolution, wherein after mixing an amount of said base composition withan amount of said peroxide solution a mouthwash composition is providedcomprising (a) an antimicrobial effective amount of thymol and one ormore essential oils; (b) optionally, ethanol in an amount up to about30% v/v, preferably up to about 22%, most preferably up to about 10%;(c) from about 0.1 to about 8.0% w/v, preferably from about 0.1 to about3.0% w/v peroxide; (d) at least one surfactant in an amount sufficientto solubilize said essential oils; and (e) water. The preferred rangesand components set forth for the mouthwash compositions of thisinvention are applicable to the calculation of the concentration of thecomponents in the base compositions and peroxide solutions of thetwo-part mouthwash system of this invention.

The first and second vessels of the two-part mouthwash system may beseparate containers or two compartments integrally united in a singlecontainer. If the vessels are separate containers, then basecompositions and peroxide solutions may be mixed in another containerprior to use simply by pouring in the predetermined ratio of the basecomposition and peroxide solution. On the other hand, if the basecomposition and peroxide solution are stored in an integrally unitedcontainer, such a container may be designed so that the base compositionand peroxide solution are mixed in an appropriate ratio as the mouthwashcomposition is dispensed from the container. Such a container mayemploy, for example, two channels, one from each vessel, which merge ina mixing chamber, and from which there is an outlet to dispense themixed mouthwash composition. Or mixing can occur in another container.Such a device could preferably employ one-way valves in the channels toavoid contamination of the base composition and peroxide solution in thevessels. Other types of vessels and containers could also be employed inthe two-part mouthwash system of this invention.

Water is added to q.s. the base composition and aqueous peroxidesolution, and the base composition and aqueous peroxide solution maythen be bottled and packaged for shipping. The mouthwash composition ofthe present invention may also be formulated, if desired, as gels, foamsor pastes, using standard formulations known in the art as appropriate.

Alternatively, the base compositions of the present invention may beformulated in a chewing gum, semi-solid (paste, gel, foam), or liquidconcentrate form. In such embodiments, for example, water is added toq.s. the volume to the necessary total for a semi-solid or liquidconcentrate.

R-Factor

Biofilms of the microorganism Streptococcus mutans (ATCC #25175) grownon stainless steel wires simulate thick, semipermeable dental plaque.For purposes of the present invention, an “R-Factor” is a convenientmeasure of the antimicrobial efficacy of the mouthwash compositions ofthe present invention, as measured by their ability to kill thosebiofilms. The R-Factor is defined as the ratio of (1) the time, inminutes, necessary for a mouthwash composition to kill S. mutansmicroorganism biofilms grown in vitro on stainless steel wires, to (2)the time, in minutes, necessary for a standard high alcohol mouthwashcomposition to kill similar biofilms of the same microorganism grown invitro on other, identical stainless steel wires. Those kill times areobtained by a plaque penetration assay developed by the presentinventors.

Plaque Penetration Assay

The plaque penetration assay employed by the present inventors to obtaintheir biofilm critical kill times is a modification of the well-knownprocedure of Tanzer, et al., described or referenced in, e.g., Tanzer,et al., “Structural requirements of Guanide, Biguanide and BisbiguanideAgents for Antiplaque Activity,” Antimicrobial Agents and Chemotherapy,December 1977, pp. 721-729; and Tanzer, et al., “In Vitro Evaluation ofSeven Cationic Detergents as Antiplaque Agents,” Antimicrobial Agentsand Chemotherapy, March 1979, pp. 408-414.

The high alcohol mouthwash composition employed by the present inventorsas their standard for their plaque penetration assay contains 27% v/vethanol and has the composition shown in the following Table 1:

TABLE 1 Ingredient Amount Ethanol (USP) 284 mls Thymol 0.639 gramEucalyptol 0.922 gram Menthol 0.425 gram Methyl Salicylate 0.64 gramBenzoic Acid 1.5 grams Caramel 0.215 gram Poloxamer 407 1.0 gram WaterQ.S. to 1 Liter

The media required for the plaque penetration assay include steriledeionized water; Letheen Broth (DIFCO); and Jordan's complex medium(with and without bromocresol purple pH indicator) [see Jordan, H. V. etal. J. Dent. Res. 39: 116-123 (1960)]. The equipment requirements forthe assay include a large number of sterile glass test tubes (e.g.,13×100 millimeters); test tube racks to hold multiple rows of sampletubes; an autoclave; and stainless steel Nichrome wires (1.5×85 mm). Itis convenient to attach each plaque wire to a Morton Closure (withoutfingers) by any suitable means, such as welding.

Jordan's medium may be prepared by blending the following ingredientswith heating as necessary:

Ingredient Amount Trypticase Peptone (BBL) 5 grams Yeast Extract 5 gramsK₂HPO₄ 5 grams Stock Salts Solution (see below) 0.5 ml Sucrose 50 gramsSodium Carbonate 0.05 grams Deionized Water Q.S to 1 Liter Stock SaltsSolution Ingredient Concentration MgSO₄(anhydrous) 3.9 g/L FeCl₃ .6H₂O0.4 g/L MnCl₂ (anhydrous) 0.12 g/L Distilled water Q.S. to 1 Liter

The pH is adjusted to 7.1 with 5N HCl followed by cooling to roomtemperature, if necessary. 50 ml is removed from the batch and placed ina 100 milliter flask. Jordan's medium with pH indicator (“recoverymedium”) is prepared by adding 1 ml of a 1% bromocresol purple stocksolution (i.e., 0.1 gram bromocresol purple in 10 mls distilled water)to 1 liter of Jordan's medium.

The culture for the assay is prepared as follows. Upon receipt, the ATCCculture is rehydrated and subcultured according to the directionssupplied by the American Type Culture Collection. The subculture isstreaked for purity on Brain-Heart Infusion Agar (DIFCO) and inoculatedinto 100 ml of sterile BHI. The agar plates are examined for purityafter 14-18 hours. If acceptable, 11 ml of sterile glycerin are added tothe BHI culture, vortexed and then subdivided into 1.8 ml cryogenictubes. The cultures are then stored at −80° C.

Four days prior to an assay, a frozen vial is thawed and added to thesmall, 100 ml flask containing 50 ml of Jordan's medium to start thecultures used for the biofilm assay. After 14-18 hours, the contents ofthe small flask are decanted aseptically into 2 liters of Jordan'smedium. The resulting inoculated medium is then aseptically dispensed,in 5.0 ml portions, into a number of empty sterile test tubes, each tubehaving a plaque wire-quipped Morton cap. The inoculated tubes are thenincubated anaerobically overnight (i.e., 14-16 hours) at 37° C.

The number of test tubes will vary depending upon the number ofdifferent mouthwash samples being tested, but it will be convenient todescribe an assay of a standard high alcohol mouthwash and four reducedalcohol mouthwash samples, which requires racks each holding 75 testtubes (i.e., five rows of fifteen tubes, each row comprising five setsof three tubes each). The first set of three tubes in each row isusually reserved for the standard high alcohol mouthwash, with thesucceeding four sets of three tubes each in that row being reserved forthe four reduced alcohol samples. There are three tubes in each setbecause each assay is performed in triplicate.

After the overnight incubation, the plaque wires are then transferredinto fresh Jordan's medium in 75 tubes (in a second rack) and againincubated anaerobically for 24 hours at 37° C. This procedure isrepeated once more. Thus, the plaque wires are cultured for 3 days, withtwo transfers after initial inoculation.

On the third day, just prior to the assay, five additional racks (eachcontaining 75 sterile test tubes) are prepared a first (assay) rackwhose test tubes each contain 6 ml of the sample mouthwashes; a second(water) rack whose test tubes each contain 6 ml of sterile deionizedwater, third and fourth racks whose test tubes each contain 6 ml ofLetheen broth rinse; and a fifth rack whose test tubes each contain 5 mlof Jordan's recovery medium. For convenience, the racks may be markedfor test series identity and time (by row).

Assay Procedure: Each assay will involve triplicate testing at timepoints separated by one minute intervals, e.g., at 2, 3, 4, 5 and 6minutes of mouthwash treatment. The first (or bottom) row of each rackcorresponds to the first test time and the succeeding four rowscorrespond, respectively to the next four test times. The exact time ofexposure of the plaque wires to the sample mouthwashes can be variedaccording to the thickness of the “plaques;” ideally, the exposureperiod will result in positive microorganism growth in the first one ortwo sampling intervals of the high alcohol control group, (i.e., thefirst and second row) and no growth thereafter. Establishing lower andupper limits of exposure required for complete kill by the controlmouthwash permits an accurate comparison of the four sample mouthwashesto this control. Mouthwash exposure takes place in a 37° C. NewBrunswick shaking water bath (shake speed 3) and may be staggered sothat the 5 time points are run concurrently, but with sufficient time topermit accurate timing and handling.

(1) To start assay, transfer one row of plaque wires to the first(bottom) row of tubes in the rack containing 6 ml of sterile water.Leave in place 2 minutes. Repeat for the next four rows of plaque wires.

(2) After the water rinse, transfer each row of plaque wires into theappropriate, corresponding, row of tubes in the rack containing 6 ml oftest mouthwash. Leave each row of plaque wires in place, with shaking inthe 37° water bath, for its treatment (exposure) period; i.e., removethe rows of plaque wires sequentially at 5 preset time points so thateach succeeding row of plaque wires is exposed to a mouthwash forsuccessively longer periods of time (e.g., 2, 3, 4, 5 and 6 minutes;individual timing can vary according to estimated “plaque” thickness.

(3) For each row of plaque wires, at the end of its treatment period,immediately remove the row and place it in the appropriate correspondingrow of the first rack of 6 ml Letheen Broth neutralization/rinse tubes.Leave each row of wires in that broth for 5 minutes and then transfer itto the appropriate corresponding row of the second rack of 6 ml LetheenBroth rinse tubes.

(4) At the end of the second Letheen Broth rinse, remove each row ofplaque wires and place it in the appropriate corresponding row of therack of 6 ml Jordan's recovery medium (with bromocresol purple).Incubate anaerobically for 48 hours at 37° C.

(5) Read for growth (+) or no growth (−) at 24 and 48 hours. Positivegrowth is indicated by a color change from purple to yellow (i.e., ifthe microorganism is still viable, it will produce an acid which causesthe color change); positive growth is often accompanied by an increasein broth turbidity.

Determination of Critical Kill Times and R-Factor: Since each mouthwashsample is located in the same set of three tubes in each row of therack, the critical time necessary for the sample to completely kill themicroorganism can be determined by observing the point (front to back orbottom to top, as the case may be) at which the Jordan recovery mediumcolor changed from yellow to purple. The critical kill time for anysample, divided by the critical kill time for the control mouthwash inthat same rack, gives the R-Factor for that sample.

Table 2 below summarizes a statistical scale developed by the presentinventors which relates the observed change from growth (+) to no growth(−) to critical kill times. For example, as shown in the first row ofTable 2, where the observed condition changes from growth (continuous+'s) to no growth (continuous −'s) (“no anomaly”), the critical killtime is determined by adding 0.50 minute to the time at which the lastgrowth observation (+) was made. The balance of Table 2 sets forth howcritical kill times are determined for different observed growth/nogrowth intervals between continuous growth segments and continuous nogrowth segments.

TABLE 2 BUSCH Scores For Critical Kill Times (CKT) Intervals betweencontinuous +‘s and −‘s Add To Last (+) Time No anomaly 0.50 −+ 1.50 −++2.90 −+++ 4.10 −+−+ 2.50 −+−−+ 2.10 −++−+ 4.06 −−+ 1.10 −−++ 2.50 −−+−+3.84 −−−+ 0.90 −−−−+ 0.80

By way of further example, consider the examples of growth/no growthsequences, and their associated critical kill times, in Table 3. In thefirst row of Table 3, there was no anomaly between continuous +'s andcontinuous −'s; therefore, CKT (per Table 2)=4.0+0.5=4.5 minutes i.e.,kill occurred somewhere between 4.0 and 5.0 minutes). In the second rowof Table 3, the interval between continuous +'s and continuous −'s is +;therefore, CKT (per Table 2)=2.0+1.5=3.5 minutes.

TABLE 3 Examples of Growth/No Growth Sequences and CKT Treatment Times(min) 2.0 3.0 4.0 5.0 6.0 CKT + + + − − 4.5 + − + − − 3.5 + + + + + >6.5− − − − − <2.0 + − − + − 3.1

In the case of rows 3 and 4 of Table 3, clearly no end point wasreached. It is assumed here that kill will occur at some point in excessof 6.5 minutes (>6.5) or much below 2.0 minutes (<2.0), respectively.

Row 5 (Table 3) is an example where the kill scale is dependent on thevalues which are located to the left of the last + and to the right ofthe first −. For that particular example, CKT=2.0+1.1=3.1 minutes (perTable 2).

The following examples are provided to illustrate the present inventionand its advantages. Throughout the examples, all percentages are statedas % w/v except where noted (e.g., ethanol contents are stated as %v/v).

EXAMPLE 1

A base composition was prepared having the following components indeionized water:

Components % w/v Thymol 0.128 Menthol 0.035 Methyl salicylate 0.128Eucalyptol 0.184 Ethanol (USP) 20.0 (v/v) Sodium Lauryl Sulfate 0.6MgSO₄ 0.6

This base composition (100 ml) was prepared in the following manner.First, 21.05 ml ethanol (USP) was transferred to a beaker and then 0.6 gsodium lauryl sulfate and 0.6 g MgSO₄ were added with stirring. Next,0.1844 g eucalyptol, 0.1278 g thymol, 0.085 g menthol and 0.128 g methylsalicylate were added to the mixture, followed by the addition ofdeionized water to bring the composition to a volume of 90 ml.Therafter, the pH was adjusted to 4.2 with 0.1N HCl, 0.1 N NaOH, or asuitable buffer e.g. sodium benzoate/benzoate acid and the compositionq.s. to 100 ml with deionized water to obtain the base composition.

EXAMPLE 2

Three peroxide solutions were prepared in deionized water having thefollowing composition:

Concentration (% w/v) Component Solution A Solution B Solution C H₂O₂2.0 4.0 5.8 Benzoic Acid 0.3 0.3 0.3

Solution A was prepared by adding 0.3 g benzoic acid to 6.67 ml of a 30%hydrogen peroxide solution in water. The solution was q.s. to 90 ml withwater and the pH adjusted to 4.2 with 0.1 N HCl or 0.1N NaOH. Afteradjusting the pH, the solution was q.s. to 100 ml with deionized waterto obtain aqueous peroxide solution A. Solutions B and C were preparedin a similar manner, except that 13.33 ml and. 19.33 ml of 30% hydrogenperoxide solution in water, respectively, were used to prepare eachsolution.

EXAMPLE 3

A peroxide containing reduced alcohol mouthwash composition (1/A) wasprepared by mixing equal volume portions of the base compositionprepared in Example 1 with the peroxide solution A prepared in Example2. Two other peroxide containing reduced alcohol mouthwash compositions1/B and 1/C, respectively) were prepared in a similar manner usingperoxide solutions B and C prepared in Example 2. The resultingcompositions are described in Table 4.

TABLE 4 Base Composition/Peroxide Solution % w/v Component 1/A 1/B 1/CThymol 0.064 0.064 0.064 Menthol 0.043 0.043 0.043 Methyl salicylate0.064 0.064 0.064 Eucalyptol 0.092 0.092 0.092 Ethanol (% v/v) 10.0 10.010.0 Sodium Lauryl Sulfate 0.3 0.3 0.3 MgSO₄ 0.3 0.3 0.3 H₂O₂ 1.0 2.02.9 Benzoic Acid 0.15 0.15 0.15 Deionized Water q.s. q.s. q.s.

EXAMPLE 4

A of base composition were prepared in a manner similar to Example 1.The concentration of the components of those compositions in deionizedwater are set forth in Table 5 below.

TABLE 5 % w/v Components 4a 4b 4c 4d 4e Thymol 0.128 0.128 0.128 0.1280.128 Menthol 0.085 0.085 0.085 0.085 0.085 Methyl Salicylate 0.1280.128 0.128 0.128 0.128 Eucalyptol 0.184 0.184 0.184 0.184 0.184 Ethanol(% v/v) 10.0 10.0 — 10.0 1.0 Sodium Lauryl Sulfate 0.6 — — — —Pluronic ® F127¹ — 0.6 0.9 — — Tauranol ® ² — — — 0.6 0.6 MgSO₄ 0.6 0.60.6 0.6 0.6 Benzoic Acid 0.3 0.3 0.3 0.3 0.3 Deionized Water q.s. q.s.q.s. q.s. q.s. ¹Pluronic ® F-127 is poloxamer 407 available from BASF²Tauranol ® is sodium-N-methyl-N-cocoyl taurate available from Finetex,New Jersey

The preparation of these base compositions is described with moreparticularity below. Base composition 4a was prepared by transferring10.53 ml of ethanol (USP) to a beaker, followed by the addition withstirring of 0.06 g sodium lauryl sulfate, 0.6 g MgSO₄ and 0.3 g benzoicacid. To this mixture was added 0.1844 g eucalyptol, 0.1278 g thymol,0.085 g menthol and 0.128 g methyl salicylate. The composition was thenq.s. to 90 ml with deionized water, the pH adjusted to 4.2 and then q.s.to 100 ml with deionized water to obtain base composition 4a. Basecomposition 4b was prepared in a similar manner, with the exception that0.6 g Pluronic® F127 was substituted for the sodium lauryl sulfate.

The base composition having no alcohol (4c) was prepared by transferring75 ml deionized water to a beaker, followed by adding with stirring 0.9g Pluronic® F127, 0.6 g MgSO₄ and 0.3 g benzoic acid. The identicalessential oils employed in base compositions 4a and 4b were then added,the solution was q.s. to 90 ml by adding dionized water, the pH adjustedto 4.2 and the solution q.s. to 100 ml with deionized water to obtainbase composition 4c.

Two base compositions containing the anionic surfactant Tauranol® wereprepared. Base composition 4d was prepared in a manner similar to basecomposition 4a with the exception that 0.6 g Tauranol® was substitutedfor sodium lauryl sulfate. Base composition 4e was prepared in a mannersimilar to base composition 4d, except that 1.05 ml of ethanol (USP) wasused instead of 10.53 ml.

EXAMPLE 5

Two peroxide solutions were prepared in deionized water having thefollowing composition:

Solution D Solution E 4.0% w/v H₂O₂ 5.8% w/v H₂O₂

Solutions D and E were prepared by adding 13.33 ml and 19.33 ml,respectively, of a 30% hydrogen peroxide solution to a beaker andbringing the total volume of each solution to 90 ml with deionizedwater. Each solution was then adjusted to a pH of 4.2 with 0.1 N HCl or0.1N NaOH and then each solution was q.s. to 100 ml by adding deionizedwater to obtain aqueous peroxide solutions D and E.

EXAMPLE 6

A series of peroxide containing reduced alcohol or alcohol-freemouthwash compositions were prepared using the base compositions forExample 4 and the peroxide solutions of Example 5. Each mouthwashcomposition was prepared by mixing equal volume amounts of a basecomposition and a peroxide solution. The resulting compositions aredescribed in Table 6.

TABLE 6 Base Composition/Peroxide Solution (% w/v) Components 4a/D 4a/E4b/D 4b/E 4c/D 4c/E 4d/D 4d/E 4d/D 4d/E Thymol 0.064 0.064 0.064 0.0640.064 0.064 0.064 0.064 0.064 0.064 Menthol 0.043 0.043 0.043 0.0430.043 0.043 0.043 0.043 0.043 0.043 Methyl Salicylate 0.064 0.064 0.0640.064 0.064 0.064 0.064 0.064 0.064 0.064 Eucalyptol 0.092 0.092 0.0920.092 0.092 0.092 0.092 0.092 0.092 0.092 Ethanol (USP) (% v/v) 5.0 5.05.0 5.0 — — 5.0 5.0 0.5 0.5 Sodium Lauryl Sulfate 0.3 0.3 — — — — — — —— Pluronic ® F127 — — 0.3 0.3 0.45 0.45 — — — — Tauranol ® — — — — — —0.3 0.3 0.3 0.3 MgSO₄ 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 BenzoicAcid 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 H₂O₂ 2.0 2.9 2.02.9 2.0 2.9 2.0 2.9 2.0 2.9

The peroxide containing reduced alcohol mouthwash compositions of thisinvention were tested for plaque penetration efficacy. Each of themouthwash compositions of Table 4 and 6 were evaluated in vitro for itsability to kill microorganisms in a biofilm, using the protocoldescribed above. The results are shown below in Table 7, which alsoshows the percentages of ethanol, H₂O₂ and surfactant in thosecompositions.

TABLE 7 Mouth- wash Average CKT Com- Ethanol H₂O₂ Surfactant (Min) Rposition % v/v % w/v % w/v Composition Control Factor 1/A 10.0  1.0 0.3(S) 5.25 3.17 1.66 1/B 10.0  2.0 0.3 (S) 3.25 3.17 1.02 1/C 10.0  2.90.3 (S) <3 3.17 <0.9 4a/D 5.0 2.0 0.3 (S) 3.25 3.92 0.83 4a/E 5.0 2.90.3 (S) 3.58 3.92 0.91 4b/D 5.0 2.0 0.3 (P) 3.25 3.35 0.97 4b/E 5.0 2.90.3 (P) 2.58 3.35 0.77 4c/D — 2.0 0.45 (P)  3.58 3.35 1.07 4c/E — 2.90.45 (P)  3.58 3.35 1.07 4d/D 5.0 2.0 0.3 (T) 3.25 4.25 0.76 4d/E 5.02.9 0.3 (T) 2.25 4.25 0.53 4e/D 0.5 2.0 0.3 (T) 3.25 4.25 0.76 4e/E 0.52.9 0.3 (T) 2.25 4.25 0.53 S—Sodium Lauryl Sulfate P—Pluronic ® F127T—Tauranol ®

The results show that mouthwash compositions of this invention evenhaving no alcohol exhibited plaque penetration activity substantiallyequivalent to the high alcohol containing control. When preferredamounts of hydrogen peroxide were used, the efficacy of the mouthwashcomposition was in some cases substantially better than that of thecontrol, even at alcohol levels as low as 0.5% v/v.

Kill Kinetics Assays

The kill kinetics of several hydrogen peroxide aqueous solutions wereassayed along with high alcohol containing control and a sterile watercontrol. Kill Kinetics assays were performed by determining the growthof microorganisms after time (0.5, 1, 2, 5, and 10 minutes) exposure totest product. Neutralizing media was inoculated and incubated for 48 to72 hours and growth/no growth recorded. The effect of biological fluidson the kill kinetics assay was determined by performing the assay withinoculum of each microorganism prepared in heat inactivated horse serum

Microorganisms

Cultures of all microorganisms were obtained from the American TypeCulture Collection (ATC), Rockville, Md., reconstituted, aliquoted andmaintained by freezing at −100° C. over liquid nitrogen. All aerobeswere cultivated a 37° C., with the exception of Candida albicans whichwas cultivated at 30° C., and all anaerobes were cultivated at 37° C.,under an anaerobic atmosphere.

Preparation of Microorganisms

The test organisms were transferred from frozen stock cultures andpassed at least 3 time in modified Schaedler's broth and maintained byserial passage. For all tests, fresh cultures in late log or earlystationary phase were used for inoculating. Faster growing cultures wereused 16-24 hours after incubation, while some slower growing speciesrequired 2-3 days of incubation. Microorganisms were incubated eitheraerobically (37° C.) or anaerobically (37° C.).

Media

Growth Media

All cultures were grown in Schaedler broth (Difco or BBL) supplementedwith 1 μg/ml soluble menadione (Sigma Chemical Co. #M5750), dispensedand sterilized by autoclaving.

Test Media

Neutralizing broth was comprised of modified Schaedler brothsupplemented with Azolectin and Tween 80. The neutralizing broth wasprepared by dissolving 28.4 grams of Schaedler broth in 900 ml of water.Soluble menadione was added to give a final concentration of 1 μg/m.Then 15 ml of the Neutralizing stock solution was added and the solutionwas q.s. to 1 liter with distilled water. The media was dispensed intotest tubes and autoclaved.

The Neutralizing stock solution was prepared as follows: 35 grams ofAzolictin (L-α-Phosphatidylcholine) was combined with 700 ml ofdistilled water. This was mixed and heated until the Azolectin wasdissolved. Tween 80 (Polysorbate 80 NF) (250) grams) and potassiumphosphate buffer (1 ml ) were added. The solution was mixed and heateduntil dissolution was complete. When the stock became clear it was q.s.to 1 liter and cooled to room temperature. The pH was adjusted to 7.2with 1 N NaOH. The solution was aliquoted into glass containers,autoclaved and stored a 4° C.

Potassium phosphate buffer was prepared by dissolving 3.74 grams ofKH₂PO₄ in 900 ml of distilled water. The pH was adjusted to 7.2 and q.s.to 1000 ml with distilled water.

Neutralizing Test

To assume adequate product neutralization a Neutralizing test wasperformed on each organism at the time of a kill kinetics assay. Eachproduct (0.1 ml) was added to 10 ml of Test Neutralizing media. Eachtube was inoculated with 0.1 ml of a 1:100 dilution of a standardinoculum of each organism in growth media. The diluted inoculum was usedwithin 15 minutes of preparation. The tubes were incubated for 48 hoursunder the appropriate growth conditions and examined for growth byvisual observation.

Kill Time Determination

Five hundred μl of inoculum of each test organism were inoculated into4.5 ml of test product. Using a stop watch to time the test, a 0.5 mlaliquot of the reaction mixture was removed at 0.5, 1, 2, 5 and 10minutes, added to 4.5 ml of fresh neutralizing test medium, mixed andincubated at 37° C. (except for C. albicans which was incubated at 30°C.) for 48 to 72 hours. After incubation the tubes were examined forgrowth by visual observation.

Inoculation was accomplished with the aid of a Matrix TechnologiesProgrammable Electrapette (supplied by Warner Lambert). Six tubes wereinoculated simultaneously. Each assay consisted of inoculating one tubeof each test product. All assays were performed three times induplicate, using a separate inoculum preparation.

Kill Time Determination—Effect of Biological Fluids on Inhibition

Heat activated horse serum (Gibco) was used to determine the effect ofbiological fluids upon antiseptic efficacy. Equal volumes of serum andeach test organism were mixed and used as the inoculum. One ml wasinoculated in 4 ml of test product. The kill time determination assaywas then performed by inoculation of test medium followed by incubationas described above.

Each assay was done three times in duplicate.

Culture Monitoring

All cultures were monitored for purity by periodic transfer to Bloodagar plates and selective media if appropriate. Cultures were checkedfor purity by visual observation of colonial morphology after anappropriate incubation period of 48 hours to 5 days.

Media Monitoring

Tubes of all media without test agent were inoculated with theappropriate organism to be assayed to serve as a positive control forgrowth. Tubes of uninoculated media from each batch of media preparedwere also incubated to serve as a negative control for sterility.

Interpretation of Results

Results were recorded as the first time the inoculated tube exhibited nogrowth (i.e., the minimum time required to completely kill the testorganisms). The summary critical kill time was recorded as the firstpoint at which three or more of the six subculture tubes exhibited nogrowth. All tests in which three tubes showed growth were repeated forconfirmation.

The results of the assay are set forth in Table 8 below.

TABLE 8 Kill Kinetics Assay w/Serum F. nucleatum S. aureus C. albicans#10953 #6538 #18804 0.5% H₂O₂ <0.5 >5.0 >5.0 1.0% H₂O₂ <0.5 <0.5 >5.01.5% H₂O₂ <0.5 <1.0 <5.0 Listerine <0.5 <1.0 <0.5 Sterilewater >5.0 >5.0 >5.0

This assay indicates the hydrogen peroxide aqueous solutions are notalone broad spectrum antiseptic and therefore the antiplaque efficacy ofa combination of hydrogen peroxide and antimicrobial effective amountsof thymol and one or more other essential oils was not predictable.

EXAMPLE 7

A series of peroxide containing reduced alcohol or alcohol-freemouthwash compositions were prepared. Each composition was prepared bymixing a solution containing the essential oils, thymol, menthol, methylsalicylate and eucalyptol, the listed surfactants and ethanol, ifpreset, with an aqueous peroxide solution. The resulting mouthwashcomposition contained 0.064% w/v thymol, 0.043% w/v menthol 0.065% w/vmethyl salicylate and 0.092% w/v eucalyptol. The concentration of theethanol, peroxide and surfactants in the mouthwash composition as wellas the plaque kill activity expressed as an R factor (as describedabove) are set forth in Table 9 below.

Plaque Kill Ethanol H₂O₂ SLS¹ F127² Activity Composition (% v/v) (% w/v)(% w/v) (% w/v) (R factor) 7A  0 0.5 0.2  1.0 >1.3 7B  0 1.0 0.2  1.01.1 7C  0 1.5 0.2  1.0 0.7 7D  0 1.5 0.25 1.0 1.0 7E  0 0.5 0.3 1.0 >1.4 7F  0 1.0 0.3  1.0 1.2 7G  0 1.5 0.3  1.0 0.8 7H  0 1.5 0.251.0 1.2 7I 10 0.5 0.20  0.20 >1.4 7J 10 1.0 0.20  0.20 1.1 7K 10 1.50.20  0.20 0.8 7L 10 1.5 0.25  0.20 0.8 ¹Sodium Lauryl Sulfate²Pluronic ® 127 is poloxamer 407 available from BASF

The results indicate that the reduced alcohol and alcohol-freecompositions containing about 1.0 to about 1.5% w/v hydrogen peroxideare highly effective antimicrobial compositions. Particularly preferredcompositions are (i) the alcohol free composition containing about 0.3%w/v sodium lauryl sulfate and about 1.0% poloxame 407, and (ii) thereduced alcohol composition containing about 0.20% w/v sodium laurylsulfate and about 0.20% w/h poloxamer 407.

What is claimed is:
 1. A peroxide-containing two-part mouthwash systemcomprising: (i) a first vessel containing a base composition comprisingan essential oil mixture comprising thymol, eucalyptol, menthol andmethylsalicylate, optionally ethanol, at least one anionic surfactantand water and (ii) a second vessel containing an aqueous peroxidesolution such that the aqueous peroxide solution is isolated fromessential oils capable of reacting with the aqueous peroxide solution,wherein the contents of the first vessel and the second vessel can bedispensed and mixed into a separate container or compartment just priorto use, forming a mouthwash solution comprising: (a) from about 0.16 toabout 0.5% of an essential oil mixture comprising: thymol, eucalyptol,menthol and methylsalicylate; (b) optionally, ethanol in an amount up toabout 30% v/v; (c) from about 0.1% to about 8.0% w/v of a peroxide; (d)a sufficient amount of at least one anionic surfactant to assist in thesolubilization of said essential oils; and (e) water.
 2. A Theperoxide-containing two-part mouthwash system of claim 1, wherein theamount of ethanol in said antimicrobial composition is no more thanabout 22% v/v.
 3. The peroxide-containing two-part mouthwash system ofclaim 1, wherein the amount of ethanol in said antimicrobial compositionis no more than about 10% v/v.
 4. The peroxide-containing two-partmouthwash system of claim 3, wherein the peroxide in said antimicrobialcomposition is hydrogen peroxide present in an amount from about 0.1% toabout 3.0% w/v.
 5. The peroxide-containing two-part mouthwash system ofclaim 4, wherein the amount of hydrogen peroxide in said antimicrobialcomposition is from about 0.1% to about 2.9% w/v.
 6. Theperoxide-containing two-part mouthwash system of claim 4, wherein thesurfactant is selected from the group consisting of non-ionicsurfactants, anionic surfactants and mixtures thereof.
 7. Theperoxide-containing two-part mouthwash system of claim 6, wherein thesurfactant is at least one non-ionic surfactant.
 8. Theperoxide-containing two-part mouthwash system of claim 7, wherein saidnon-ionic surfactant is selected from the group consisting ofpoloxamers.
 9. The peroxide-containing two-part mouthwash system ofclaim 8, wherein the amount of poloxamer in said antimicrobialcomposition is from about 0.01% to about 1.0% w/v.
 10. Theperoxide-containing two-part mouthwash system of claim 9, wherein saidpoloxamer is Poloxamer
 407. 11. The peroxide-containing two-partmouthwash system of claim 6, wherein the surfacant is at least oneanionic surfactant.
 12. The peroxide-containing two-part mouthwashsystem of claim 11, wherein said anionic surfactant is selected from thegroup consisting of sodium lauryl sulfate, magnesium lauryl sulfate andsodium N-methyl-N-cocoyl taurate.
 13. The peroxide-containing two-partmouthwash system of claim 12, wherein the amount of said anionicsurfactant in said antimicrobial composition is from about 0.01% toabout 1.0% w/v.
 14. The peroxide-containing two-part mouthwash system ofclaim 1, wherein an amount of thymol in said antimicrobial compositionis from about 0.001% to about 0.5% w/v.
 15. The peroxide-containingtwo-part mouthwash system of claim 14, wherein said antimicrobialcomposition contains eucalyptol in an amount of from about 0.001% toabout 0.5% w/v, menthol in an amount of from about 0.001% to about 0.5%w/v, and methyl salicylate in an amount of from about 0.001% to about0.5% w/v.
 16. The peroxide-containing two-part mouthwash system of claim1, wherein the amount of said base composition to be mixed with theamount of said peroxide solution is a ratio of about 1:10 to about 10:1.17. The peroxide-containing two-part mouthwash system of claim 16,wherein the ratio of base composition to peroxide solution in saidantimicrobial composition is about 1:1.
 18. The peroxide-containingtwo-part mouthwash system of claim 1, wherein said first vessel and saidsecond vessel are integrally united.
 19. The peroxide-containingtwo-part mouthwash system of claim 1, wherein an R-Factor of saidantimicrobial composition is less than about 1.2.
 20. Theperoxide-containing two-part mouthwash system of claim 1, wherein anR-Factor of said antimicrobial composition is less than about 1.0.